Viral vectors having enhanced effectiveness with reduced virulence

ABSTRACT

The present invention provides methods of use of recombinant vaccinia virus from which the region encoding the N-terminal 83 or 54 amino acids of the E3L gene product has been deleted, or amino acids at positions 44 and 66 have been mutated. Compositions comprising the recombinant vaccinia virus are also provided.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of PCT/US00/10948 whichapplication was published by the International Bureau in English on Dec.7, 2000 and which claims priority of U.S. application Ser. No.60/136,277 filed May 27, 1999. The disclosures of PCT/US00/10948 andSer. 60/136,277 are incorporated herein by reference in theirentireties.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

Financial assistance for this project was provided by the U.S.Government through the National Institutes of Health under grant numberCA-4865409 and the United States Government may own certain rights inthis invention.

BACKGROUND OF THE INVENTION

Vaccinia virus is a member of the poxvirus family of DNA viruses.Poxviruses including vaccinia virus are extensively used as expressionvectors since the recombinant viruses are relatively easy to isolate,have a wide host range, and can accommodate large amounts of DNA.

The vaccinia virus genome contains nonessential regions into whichexogenous DNA can be incorporated. Exogenous DNA can be inserted intothe vaccinia virus genome by well-known methods of homologousrecombination. The resulting recombinant vaccinia viruses are useful asvaccines and anticancer agents.

The use of vaccinia virus recombinants as expression vectors andparticularly as vaccines and anticancer agents raises safetyconsiderations associated with introducing live recombinant viruses intothe environment. Virulence of vaccinia virus recombinants in a varietyof host systems has been attenuated by the deletion or inactivation ofcertain vaccinia virus genes that are nonessential for virus growth.However, there remains a need in the art for the development of vectorsthat have reduced pathogenicity while maintaining desirable propertiesof wild-type virus, such as host range, and active protein synthesis ofa desired gene product.

SUMMARY OF THE INVENTION

The present invention provides methods of use of a recombinant vacciniavirus in which the region encoding an N-terminal portion of the E3L geneproduct has been mutated. In a preferred embodiment, the region encodingthe N-terminal 54 or 83 amino acids of the E3L gene product has beendeleted, or the amino acids at positions 44 and 66 have been mutated.The present invention further provides an expression vector comprisingthe recombinant vaccinia virus of the invention and exogenous DNA.

The present invention also provides a composition comprising theexpression vector of the invention and a carrier and a method of makinga recombinant gene product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing percent weight change in mice infected withvaccina virus following immunization with WR Δ83N.

FIG. 2 is a graph showing percent weight change in mice infected withvaccinia virus following immunization with WR Δ54N.

FIG. 3 is a graph showing percent weight change in mice infected withvaccina virus following immunization with WR N44A W66L.

DETAILED DESCRIPTION OF THE INVENTION

The vaccinia virus E3L gene codes for double-stranded RNA bindingproteins, and has been shown to be necessary for the vaccinia virusinterferon-resistant phenotype. The E3L gene product of the vacciniavirus is a 190 amino acid polypeptide. Amino acids 118 to 190 have beenimplicated in dsRNA binding, as disclosed by Kibler et al. (1997) J.Virol. 71: 1992, incorporated herein by reference.

The present invention provides a recombinant vaccinia virus in which theregion of the viral genome encoding an N-terminal portion of the E3Lgene product has been mutated. An N-terminal portion of the E3L geneproduct is defined herein as including at least amino acids 1 through 37of the E3L gene product. Amino acid numbering as used herein is adoptedfrom Goebel et al. (1990) Virology 179: 247-66, 577-63, the disclosureof which is incorporated herein by reference. An N-terminal portion ofthe E3L gene product as defined herein may encompass the region from theN-terminus (amino acid 1) up to and including amino acid 117.Accordingly, a mutation is present in the region encoding at least 37,and as many as 117, N-terminal amino acids of the E3L gene product inthe recombinant vaccinia virus of the present invention.

The term mutation, as used herein, includes deletions, substitutions andpoint mutations.

In a preferred embodiment, the region of the viral genome encoding theN-terminal 83 amino acids of the E3L gene product has been deleted. Inthis preferred embodiment, the recombinant vaccinia virus of the presentinvention contains a nucleic acid fragment encoding amino acids 84-190of the E3L gene product instead of the gene encoding amino acids 1-190of the E3L gene product at the E3L locus of the WR strain of vacciniavirus and is designated WR Δ83N.

In another preferred embodiment, the region of the viral genome encodingthe N-terminal 54 amino acids of the E3L gene product has been deleted.In this preferred embodiment, the recombinant vaccinia virus of thepresent invention contains a nucleic acid fragment encoding amino acids55-190 of the E3L gene product instead of the gene encoding amino acids1-190 of the E3L gene product at the E3L locus of the WR strain ofvaccinia virus and is designated WRΔ54N.

In another preferred embodiment, the region of the viral genome encodingthe N-terminal portion of the E3L gene product at the E3L locus of theWR strain of vaccinia virus contains two point mutations such that theamino acid at position 44 is changed from asparagine to alanine, and theamino acid at position 66 is changed from tryptophan to leucine, and isdesignated WR N44A W66L.

The present invention further provides recombinant vaccinia viralvectors comprising the recombinant vaccinia virus described above andfurther containing exogenous, i.e., nonvaccinia virus, DNA. ExogenousDNA may encode any desired product, including for example, an antigen,an anticancer agent, or a marker or reporter gene product. Therecombinant vaccinia virus may further have deletions or inactivationsof nonessential virus-encoded gene functions. Nonessential genefunctions are those which are not required for viral replication in ahost cell. The exogenous DNA is preferably operably linked to regulatoryelements that control expression thereof. The regulatory elements arepreferably derived from vaccinia virus.

The recombinant vaccinia virus of the present invention may beconstructed by methods known in the art, for example by homologousrecombination or site directed mutagenesis. Standard homologousrecombination techniques utilize transfection with DNA fragments orplasmids containing sequences homologous to viral DNA, and infectionwith wild-type or recombinant vaccinia virus, to achieve recombinationin infected cells. Conventional marker rescue techniques may be used toidentify recombinant vaccinia virus. Representative methods forproduction of recombinant vaccinia virus by homologous recombination aredisclosed by Piccini et al. (1987) Methods in Enzymology 153:545, thedisclosure of which is incorporated herein by reference. Representativemethods for site-directed mutagenesis are disclosed by Sarkar (1990)Biotechniques 8:404, the disclosure of which is incorporated herein byreference.

For example, the recombinant vaccinia virus of a preferred embodiment ofthe present invention may be constructed by infecting host cells withvaccinia virus from which the E3L gene has been deleted, andtransfecting the host cells with a plasmid containing a nucleic acidencoding amino acids 84-190 or 55-190 of the E3L gene product flanked bysequences homologous to the left and right arms that flank the vacciniavirus E3L gene. The vaccinia virus used for preparing the recombinantvaccinia virus of the invention may be a naturally occurring orengineered strain. Strains useful as human and veterinary vaccines areparticularly preferred and are well-known and commercially available.Such strains include Wyeth, Lister, WR, and engineered deletion mutantsof Copenhagen such as those disclosed in U.S. Pat. No. 5,762,938, whichis incorporated herein by reference. Recombination plasmids may be madeby standard methods known in the art. The nucleic acid sequences of thevaccinia virus E3L gene and the left and right flanking arms arewell-known in the art, and may be found for example, in Earl et al.(1993) in Genetic Maps: locus maps of complex genomes, O'Brien, ed.,Cold Spring Harbor Laboratory Press, 1.157 the disclosure of which isincorporated by reference, and Goebel et al. (1990), supra. The aminoacid numbering used herein is adopted from Goebel et al. (1990), supra.The vaccinia virus used for recombination may contain other deletions,inactivations, or exogenous DNA as described hereinabove.

Following infection and transfection, recombinants can be identified byselection for the presence or absence of markers on the vaccinia virusand plasmid. Recombinant vaccinia virus may be extracted from the hostcells by standard methods, for example by rounds of freezing andthawing.

The resulting recombinant vaccinia virus may be further modified byhomologous recombination or site directed mutagenesis to provide otherdeletions, inactivations, or to insert exogenous DNA.

It has been discovered in accordance with the present invention thatrecombinant vaccinia viruses having a mutation in the DNA encoding aN-terminal portion of the E3L gene product, and preferably a deletion ofamino acids 1-83 or 1-54 of the E3L gene product, or a point mutationsresulting in alanines at positions 44 and 66, maintain viralreplication, protein synthesis and interferon-resistance that isindistinguishable from wild-type virus, but have remarkably reducedpathogenicity in mice relative to wild-type vaccinia virus of the samestrain. Further, immunization of mice with the recombinant viruses ofthe present invention protects mice from infection with wild type virus.

The present invention further provides a composition comprising therecombinant vaccinia virus of the invention and a carrier. Also providedis a composition comprising the recombinant vaccinia viral expressionvector of the invention and a carrier. The term carrier as used hereinincludes any and all solvents, diluents, dispersion media, antibacterialand antifungal agents, microcapsules, liposomes, cationic lipidcarriers, isotonic and absorption delaying agents, and the like.

The recombinant vaccinia viruses and compositions of the presentinvention may be used as expression vectors in vitro for the productionof recombinant gene products, or as delivery systems for gene products,as human or veterinary vaccines, or anticancer agents. Such utilitiesfor recombinant vaccinia viruses are known in the art, and disclosed forexample by Moss (1996) “Poxviridae: The Viruses and Their Replication”in Virology, Fields et al., eds., Lippincott-Raven, Philadelphia, pp.2637-2671, incorporated herein by reference.

The present invention further provides a method of making a recombinantgene product comprising subjecting a recombinant vaccinia viral vectorcomprising a vaccinia virus having a mutation in the region encodingamino acids 1-117 of the E3L gene product and further comprisingexogenous DNA that encodes the recombinant gene product operably linkedto regulatory elements that control expression thereof, to conditionswhereby said recombinant gene product is expressed, and optionallyrecovering the recombinant gene product. In a preferred embodiment, therecombinant gene product is an antigen that induces an antigenic and/orimmunogenic response when the gene product or a vector that expresses itis administered to a mammal.

All references cited herein are incorporated in their entirety.

The following nonlimiting examples serve to further illustrate theinvention.

EXAMPLE 1 Construction of Recombinant Vaccinia Virus

The plasmid pMPE3ΔGPT (described by Kibler et al. (1997) J. Virol.71:1992, incorporated herein by reference) was used for recombining atruncated E3L gene into the E3L locus of the WR strain of vacciniavirus. The recombination plasmid pMPE3ΔGPT is a derivative of pBSIISK(Stratagene, La Jolla, Calif.) that has had the β-galactosidasesequences deleted, and that contains sequences homologous to the leftand right arms flanking the vaccinia virus E3L gene, but that lacks theE3L gene itself. The recombination plasmid contains the E. coli gpt geneoutside the E3L flanking arms and thus allows for selection oftransfected cells by treatment with mycophenolic acid (MPA).

The Aat II (blunt-ended) Sal I fragment of E3L was subcloned into thepGEM3-5T vector (described by Chang et al. (1993) Virology 194: 537, thedisclosure of which is incorporated by reference) and subsequentlycloned into the pMPE3ΔGPT recombination plasmid using Bam HI and HindIII restriction sites. The E3L fragment encodes amino acids 84-190 ofthe E3L gene product as numbered by Goebel et al (1990), supra, and hasa deletion of the DNA encoding the N-terminal amino acids 1-83. Theplasmid resulting from the cloning of the E3L fragment into pMPE3ΔGPT isdesignated pMP-Δ83N.

Plasmid containing Δ54N was constructed by whole-plasmid PCR usingpMPE-3L as a template and constructing primers designed to make theappropriate deletion. Briefly, divergent primers were designed flankingthe region to be deleted. The primers were phosphorylated on their 5′ends using T4 polynucleotide kinase (Gibco, BRL) as follows. Separatereactions were performed for each primer, containing 3 μl of primer at50 pmol/μl, 2 units of kinase, and 1× kinase buffer (Gibco, BRL), in afinal volume of 5μl and was placed at 37° C. for 25 min. 1.5 μl of eachof the phosphorylated primers (50-100 pmol per reaction) were added tothe following PCR reaction: 10 ng template DNA, 5 μl of 2.5 mM dNTPs(Promega), 5 μl of 10× Pfu buffer (Stratagene), and 1 unit Pfupolymerase (Stratagene). Sterile glass distilled water was added toreach a final volume of 50 μl. Controls were run without primers orpolymerase to easily determine background levels of template DNA. Thethermal cycle was programmed as follows: 94° C. for 4 min, then 16cycles of (94° C. for 1 min, 50° C. for 1 min, 72° C. for 12 min).pMPE3LN44A/W66L (pMPE3L-N/W) was provided by Alan Herbert of theMassachusetts Institute of Technology.

In vivo recombination was performed in baby hamster kidney (BHK) cells.Subconfluent BHK cells were simultaneously infected with the WR strainof vaccinia virus deleted of the E3L gene (WRΔE3L) at a multiplicity ofinfection (MOI) of 5 and transfected with 1 g of pMP-Δ83N, pMP-Δ54N orpMP-N44A/W66L using Lipofectace (Gibco BRL). WRΔE3L was prepared byreplacing the E3L gene from the WR strain of vaccinia virus with thelacZ gene, by homologous recombination with pMPE3ΔGPT in which the lacZgene was inserted between the E3L flanking arms.

Thirty hours post infection, the cells were harvested and recombinantvirus was subjected to selection as follows. Virus was extracted frominfected/transfected cells by three rounds of freezing and thawing andused to infect confluent BHK cells that had been pretreated for sixhours with MPA selection medium (Modified Eagle Medium (MEM) containing10% fetal bovine serum (FBS), 10 g/ml mycophenolic acid, 250 g/mlxanthine, 15 g/ml hypoxanthine). Following infection, cells wereoverlayed with MPA selection medium. At 24-72 hours post infection,plaques were visible and dishes were overlayed with MPA selection mediumcontaining 0.5% molten agarose and 0.4 g/ml X-gal(5-bromo-4-chloro-3-indolyl-β-galactoside). Blue plaques were isolatedfour to six hours after X-gal overlay. Two more rounds of MPA selectionwere performed on the isolated blue plaques.

Resolution of the in vivo recombination occurs when the MPA selectionmedium is removed, resulting in either recovery of the original virus,WRΔE3L (containing lacZ in the E3L locus) or a recombinant viruscontaining the Δ83N or Δ54N deletion of E3L or N44A/W66L in the E3Llocus. MPA-resistant blue plaques were used to infect untreated rabbitkidney RK13 cells. At 24-48 hours post infection, dishes were overlayedwith MEM medium containing 0.5% molten agarose and 0.4 g/ml X-gal. Bothblue and clear plaques were visible. Blue plaques indicate resolution ofWRΔE3L with lacZ in the E3L locus. Clear plaques indicate resolution ofvirus containing the Δ83N deletion of E3L in the E3L locus.

Two more rounds of infections with clear plaques were performed topurify plaques containing the desired mutation. Recombinant virus wasamplified in RK13 cells.

Nucleic acid sequencing was used to confirm that the Δ83N fragment ofE3L of plasmid pMP-Δ83N, the Δ54N fragment of E3L of plasmid pMPΔ54N orthe N44A/W66L mutation of E3L of plasmid pMP-N44A/W66L was present inthe recombinant virus. Viral DNA was extracted from cells infected byeach virus. Infected cells were freeze-thawed three times, followed by athirty second sonication. Cell debris was removed by centrifugation at700×g for ten minutes. Nucleic acid was obtained by phenol/chloroformextraction of the supernatant, and PCR was performed using primers tothe E3L flanking arms. The PCR reaction products were subjected toagarose gel electrophoresis. DNA was extracted from the band of interestand DNA sequencing was performed. The identity of the insert wasdetermined by sequence comparison to the plasmid DNA sequence ofpMP-Δ83N, pMP-Δ54N or pMP-N44A/W66L.

EXAMPLE 2 Host Range and Interferon Resistance of WR, WRΔ83N and WRΔ3L

Wild-type vaccinia virus of the WR strain (WR) and variants WRΔE3L andWRΔ83N as described in Example 1 were assessed for interferon resistanceas follows.

RK13 cells were set down in six well tissue culture dishes at 70-80%confluency. Cells were treated with varying concentrations of rabbitinterferon alpha (0-1000 U/ml) for sixteen hours prior to infection.Cells were infected with approximately 100 plaque forming units (pfu) ofWR, WRΔE3L or WRΔ83N virus. Dishes were stained with crystal violet 24hours post infection and plaques were counted.

WRΔE3L exhibited interferon sensitivity (as measured by plaquereduction) at a concentration of 10 Units/ml of interferon, whereasWRΔ83N and WR did not exhibit interferon sensitivity at 10 or 100Units/ml, but both showed plaque reduction at a concentration of 1000Units/ml.

The foregoing results indicate that WRΔE3L is sensitive to the effectsof interferon, and that WRΔ83N, like WR, exhibits aninterferon-resistant phenotype.

WR, WRΔE3L and WRΔ83N were assayed for host range as follows. Six-welltissue culture dishes containing RK13 cells or HeLa cells were set downsimultaneously at 70-80% confluency. Both cell types were infected withequal dilutions of virus, and 24-48 hours post infection cells werestained with crystal violet and plaques were counted for each cell type.A comparison was made by determining the efficiency of plaquing (numberof plaques in HeLa cells divided by number of plaques in RK13 cells) foreach virus. The efficiencies of plaquing were: WR: 0.98; WRΔ83N: 1.06;WRΔE3L: <0.01.

These results indicated that WRΔE3L has a restricted host range in thatit cannot replicate in HeLa cells but exhibits nearly wild-typereplication in RK13 cells. WRΔ83N, like wild-type WR, replicates in RK13cells and HeLa cells.

The foregoing results showthat WR and WRΔ83N are identical with respectto host range and interferon resistance in the cultured cells evaluated,whereas WRΔE3L is sensitive to interferon and has a restricted hostrange.

EXAMPLE 3 Virulence of WR, WRΔE3L and WRΔ83N

Virus (WR, WRΔE3L or WRΔ83N) was amplified by infection of RK13 cellsuntil 100% CPE (cytopathic effect) was observed. Cells were scraped andresuspended in 1 mM Tris, pH 8.8. Amplified virus was freeze-thawedthree times to release virus from cells. Debris was removed bycentrifugation at 700×g for 10 min. Supernatant was used for mouseinfections. Various dilutions of virus in 1 mM Tris, pH 8.8 were used inthe experiment to determine LD50.

Three to four week old c57b16 mice were anesthetized by intrafemoralinjection of a cocktail of ketamine, acepromazine, and xylazine. Micewere subsequently infected with 10 l of virus or a dilution of virusintranasally using a pipetman and gel loading tip. Mice were thenreplaced in their cages and observed daily for pathogenesis and death.

Intranasal inoculation with WR resulted in death at 10⁴ pfu, whereas nopathogenesis could be detected with WRΔE3L at the highest dose. Forinoculation with WRΔ83N, 10⁷ pfu was required for death, indicating thatthe amino-terminus of E3L is an important determinant for virusvirulence.

EXAMPLE 4 Vaccination with WR Δ83N, WR Δ54N and WR N44A W66L

Groups of five C54b16 mice were immunized with different doses (rangingfrom 15 to 15,000 plaque forming units) of recombinant vaccinia virusdeleted of the 83 N terminal amino acids in the E3L gene (WR Δ83N). Onemonth later the immunized mice and the unimmunized controls (mock+wt WR)were challenged with a million pfu of wt WR. Weight loss was used as anindicator of disease due to wt WR. As shown in FIG. 1, severe weightloss was observed in the unimmunized control while all the immunizedmice recorded normal weight gain following challenge. 15000 pfu of therecombinant virus was sufficient to protect mice against infection withwt WR.

Groups of five C57b16 mice were immunized with different doses (rangingfrom 20 to 20,000 plaque forming units) of recombinant vaccinia virusdeleted of the 54 N terminal amino acids in the E3L gene (WR Δ54N). Onemonth later the immunized mice and the unimmunized controls (mock+wt WR)were challenged with a million pfu of wt WR. Weight loss was used as anindicator of disease due to wt WR. As shown in FIG. 2, severe weightloss was observed in the unimmunized control while all the immunizedmice recorded normal weight gain following challenge. 20000 pfu of therecombinant virus was sufficient to protect mice against infection withwt WR.

Groups of five C57b16 mice were immunized with different doses (rangingfrom 30 to 30,000 plaque forming units) of recombinant vaccinia viruswith 2 point mutations in the N terminus in the E3L gene (WR N44A W66L).One month later the immunized mice and the unimmunized controls (mock+wtWR) were challenged with a million pfu of wt WR. Weight loss was used asan indicator of disease due to wt WR. As shown in FIG. 3, severe weightloss was observed in the unimmunized control while all the immunizedmice recorded normal weight gain following challenge. 300 pfu of therecombinant virus was sufficient to protect mice against infection withwt WR.

1. An expression vector comprising: a recombinant vaccinia virus havingan E3L gene (a) having a deletion of the region encoding amino acids1-54 of the E3L gene product and (b) encoding a protein that bindsdsRNA; and exogenous DNA and regulatory elements operably linkedthereto.
 2. A composition comprising the vector of claim 1 and acarrier.
 3. A method of making a recombinant gene product comprising:subjecting an expression vector comprising: a recombinant vaccinia virushaving an E3L gene (a) having a deletion of the region encoding aminoacids 1-54 of the E3L gene product and (b) encoding a protein that bindsdsRNA; and exogenous DNA that encodes said recombinant gene productregulatory elements operably linked thereto, to conditions whereby saidrecombinant gene product is expressed.
 4. The method of claim 3 furthercomprising recovering said recombinant gene product.
 5. The method ofclaim 3 wherein said recombinant gene product is an antigen.